Effect of Extraction Method of Chia Seeds Oil on its Content of Fatty Acids and Antioxidants

International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056
Volume: 04 Issue: 10 | Oct -2017 www.irjet.net p-ISSN: 2395-0072
© 2017, IRJET | Impact Factor value: 5.181 | ISO 9001:2008 Certified Journal | Page 545
Effect of extraction method of Chia seeds Oil on its content of fatty acids
and antioxidants
Ahmed Saad Noshe1, Aswan Hamdullah Al-Bayyar2
1,2 Food Science Dep. / College of Agriculture/ Baghdad University
---------------------------------------------------------------------------***--------------------------------------------------------------------------
Abstract-The extracted oil from the seeds of the Salvia
hispanica has been studied by different extraction methods
to obtain the highest percentage of extracted oil from white
and black chia seeds. Three extraction methods were used:
solvent extraction (cold extraction), extraction using Soxhlet
device (hot extraction), and extraction by using the screw
pressing, the study concluded that solvent extraction is the
best method for extraction in terms of quantity and quality
of extracted oil. Fatty acids were also diagnosed in each type
of oil extracted using Gas Chromatography (GC) to
determine the type of fatty acids that make up the highest
percentage of extracted oil, Where it found that the
saturated fatty acid Arachidic and unsaturated fatty acid
linoleic (omega-6) have formed the highest percentage of
the extract from the white seed oil where their percentages
(6.61%) and (3.54%), respectively, while the fatty acid
linoleic (omiga-6) of oil extracted from black seeds was
(4%). The vitamins dissolved in extracted oil from the Chia
seeds were diagnosed using high performance liquid
chromatography (HPLC). A qualitative examination was
conducted to diagnose some of the active compounds in the
extracted oil using two types of reagents for each compound,
where the presence of flavonoids, terpenes and tannins was
diagnosed. Extracted oil showed inhibition of protein
coagulation as an indicator of anti-inflammatory action.
The percentage of inhibition in oil extracted from white chia
seeds was 11.8% and 38% for oil extracted from black chia
seeds. The efficacy of the oil extracted as an antioxidant
compared to ascorbic acid was estimated as a comparative
model where it showed that the extract from black chia
seeds was the highest oil efficacy, which amounted to
(94.5%) at a concentration of 400 µl / ml compared with oil
extracted from white chia seeds which was (88.37%) at the
same concentration.
Research paper sited from the master thesis of the
first researcher.
Key words: Salvia hispanica, oil, Chia seeds, fatty acids,
antioxidants, Extraction.
1. INTRODUCTION
Seeds of Salvia Hispanica are the seeds of the Lamiaceae
and Chia seeds were used as food early in the year (3500
BC) and gained importance as a crop between 900 BC and
1500 BC in central Mexico. These seeds have been known
from the medical and nutritional point of view since
ancient times because of their high content of omega-3
fatty acids. Chia seeds have been described as a good
source of oil, protein, dietary fiber, minerals and
polyphenols [4]; [5]; [20]. Chia seed oil is a rich source of
polyunsaturated fatty acids (PUFA). Chia seed oil is unique
because it contains the highest percentage of omega-3
(ALA) more than any known natural source [3]; [6].
Omiga-3 plays an important role in health and is used in
many foods and cosmetics. Several studies have shown
that regular consumption of omega-3 has many health
effects, including prevention of cardiovascular disease,
high blood pressure and inflammatory diseases [1]; [8]. In
addition, Chia seeds and oil extracted from Chia seeds
contain a rich array of natural antioxidants such as
tocopherol, phytosterol and carotene [2]; [10], as well as
phenolic compounds including chlorogenic and kefic acid
[5], which protect consumers from many diseases and
promote human health [15].
The aim of this study was to extract the oil from the chia
seeds using three different methods to determine which
method yields the highest percentage of extracted oil and
then to detect the fatty acids found in the extracted oil by
(GC) Gas Chromatography, the diagnosis of the active
compounds in the extracted oil and study the effect of
extracted oil as an antagonist for oxidation.
2. Materials and method
2.1 Seeds
The commercial chia seeds used in this study were
obtained from Cozmo, where the source of these seeds is
Australia. The seeds are clean and homogeneous and
packed in sealed plastic containers and stored at 15-20°C.
The oil was then extracted from the seeds by solvents and

by soxhlet (hot extraction) and the use of solenoid screw
pressing.
2.2 Solvent extraction
The oil was extracted from the chia seeds using the cold
method to avoid exposure to any heat treatment using the
solvent and the method used in [10] with some
modifications in solvent ratios. Using the coffee grinder for
60 seconds, the crushed seeds were placed in a 500 mL
glass baker and 300 mL of the hexane solvent was added
(1: 3). The extraction was carried out for two consecutive
times to ensure extraction of all the oil in the seed. The
solvent was discarded using rotary evaporator at
temperature 35°C, then collect the oil extracted in the
tubes The weight of the glass tubes with the oil to
determine the percentage of oil extracted from 100 g
seeds, keeping the extracted oil under study at a
temperature of (15-20 mm) for chemical and physical
tests.
2.3 Soxhlet extraction
The percentage of oil in the Chia seeds was estimated
using the Soxhlet [23] by using hexane as a solvent for
extraction. A sample of 100 gm chia seeds was then
grinded with a coffee mill for 60 seconds and then placed
in thimble, and the weight of the beaker was recorded
before and after extraction.
2.4 Screw pressing extraction
The oil was extracted from the white and black chia seeds
using the solenoid method according to the method
mentioned in [10], with some modifications in the
extraction method. The extraction process was done at
room temperature with 100 gm of chia seeds, through a
small opening at the top of the device and was received oil
in a container placed under the device was calculated the
proportion of oil extracted through the placement in the
pipe weight information and then the weight of the tubes
with oil extracted.
2.5 Determination of total fatty acids
The total fatty acids in the extracted chia oil were
estimated as mentioned in [14] using GC chromatography
with some modifications in sample preparation rates and
sample injection conditions. The sample was prepared by
taking 0.5 ml of oil extract is added to 0.5 mL of methyl
potassium hydroxide (Methanol+ KOH) and then 5 mL of
hexane solvent is added with the stirring for a minute. This
results in two layers of the upper layer hexane with the
fatty acid (FAME) ester and the lower layer methyl
potassium hydroxide, 1 microliter of the top layer to be
injected into the device.
The conditions of the injection were as follows:
- Silica column dimensions: 60 m * 0.25 mm * 0.25
microliters
- Gas used: Hydrogen
- Gas flow rate: 1.5 ml / min
- Column temperature: 60 ° C for 2 minutes to 200 ° C at 10
° / min and then raise to 240 ° C at 7 ° / minute and then
set for 7 minutes
- Injection temperature: 270 ° C
- Detector Type: FID
- Sample size used: 1 microliter
2.6 Specific detection of some active compounds
1. Detection of terpenes
The method [9] was followed to detect turbines using the
following reagents:
Drops of chloroform and concentrated sulfuric acid were
added sequentially to 1 ml of extracted oil. The appearance
of a brownish-red color indicates the positive detection.
Anas dehydide detector: consists of the lance dehydide
detector, acetic acid and sulfuric acid. Several drops of the
anhydide dehydrogen detector were added to 1 mL of oil
extracted in the hour bottle.
2. Detection of tannins
The method used in [9] was followed to prepare each of
the following reagents, and then added to the extracted oil
to confirm the presence of tannins and as follows:
Ferric Chloride Reagent: Consists of 1 g ferric chloride in
100 ml of distilled water. Droplets of ferric chloride
reagent were added to 1 ml of extracted oil, as the
appearance of a bluish-green color indicates a positive
detection.
Lead acetate reagent: consists of 1 g lead acetate in 100 ml
distilled water. Several drops of lead acetate reagent were
added to 1 mL of extracted oil, and the appearance of a
white-yellow precipitate indicated a positive detection.

3. Detection of flavonoids
The method of [9] was followed to detect the presence of
flavonoids in the extracted oil as follows:
Magnesium and hydrochloric acid detectors: Magnesium
crystals were added to the extracted oil in an hourglass
and then added to the drops of concentrated hydrochloric
acid, the appearance of a red-orange color indicates a
positive detection.
Sulfuric acid reagent: Add drops of concentrated sulfuric
acid to the extracted oil, as the appearance of red color
indicates positive detection.
2.7 Determination of antioxidant efficacy
The anti-oxidant effectiveness of extracted chia oil was
tested for each type using the free stable root of the DPPH.
The measurements were made using the
spectrophotometer, 517 nm wavelength and Ascorbic Acid
as a positive comparison [18].
As follows:
1- A solution (0.1 mMol) of DPPH was prepared by
dissolving (1.97 mg) of the substance in (50 ml) of
absolute methanol.
2- Measuring anti-oxidant effectiveness:
The free radicals were extracted from the chia seed and
each type (white and black) by calculating the uptake of
the DPPH solution at 517 nm wavelength. The absorbance
was measured by mixing 1 ml of DPPH solution at 0.1 mM
(20, 40, 60, 80, 100) mg / L for the positive comparison of
the ascorbic acid (25, 50, 100, 200, 400) Acid.
The samples were then measured in the incubator at 37 ° C
for 30 minutes. The models were then measured in the
Spectrophotometer spectroscopy at a wavelength of 517
nm and the absorbance was converted to the percentage of
the electronic absorbent using the following equation:
% antioxidant activity = (absorbance for B – absorbance
for A) / (absorbance for A) × 100
A = Control Comparative Absorption (Control)
B = Absorption of the model (Chia oil with DPPH solution)
3. Results and discussion
3.1 Ratio of extracted oil from Chia seeds using
three methods of extraction
Table (1) shows the percentage of extracted oil from white
and black chia seeds where the percentage of oil extracted
by soxhlet (hot extraction) was 35.6% and 34.6%
respectively. This result is similar, according to [13] where
it was 35.13% and lower than that reported in [16] which
is 30.22% and less than in [7] where 30.1%. The
percentage of oil extracted by solvents (cold extraction)
was 30% for each type of seed and this is consistent with
what [21], where he pointed out that the proportion of fat
was 30% while the percentage of oil extracted by screw
pressing was 9% and 9.5%, respectively, and this result is
less than what mentioned by [10], where he pointed out
that the proportion of fat was 30%. The difference in fat
ratio in Chia seeds can be explained by the use of the three
extraction methods of the extracted chia seeds to the
different efficiency of the extraction process. The extracted
oil ratio is approximated when using the coarse extraction
method and when solvent extraction is used, In order to
avoid exposing the oil to any thermal treatment and thus
maintain its components from damage while the method of
extraction using the screw pressing gave the lowest
proportion of oil and can be due to the inefficiency of the
extraction device.
Table-1: Oil ratios of Chia seeds using three extraction
methods
Extraction
method
Oil from white
chia seed %
Oil from white chia
seed %
The Soxhlet
Method
6.536.53
Solvent method6363
Screw pressing995.
3.2 Total fatty acid
Table (2) and Table (3) show the ratio of triglyceride fatty
acids in white and black chia seed oil, where the time of
appearance of the compound was compared in the model
to be diagnosed with time of occurrence of the standard
compound Figure (1) (2) (3) By observing fatty acids, we
found that saturated arachidic acid and linoleic fatty acid
(omega-6) had the highest percentage of oil extracted from
white seeds, with a percentage of 6.61% and 3.54%,
whereas the ratio of linoleic acid to extracted oil of black

seed 4%. In comparison of the fatty acids of the oil
extracted in this study with the results of other
researchers found that the arachidic fatty acid had the
highest proportion while the proportion of linoleic in the
extracted oil from white and black seeds has formed a low
proportion, these two fatty acids are 0.15% (Arachidic)
and 20.4% (Linoleic), respectively, according to [13],[19]
showed that the ratio of these fatty acids was 0.3% and
18.1%, respectively, while the percentage of fatty acids for
oil in this study was not consistent with what was stated in
[16]. The percentage of arachidic acid was 0.29% and the
proportion of linoleic acid was 18.23%, and by observation
of the table we find that the proportion of saturated fatty
acid meristic from the oil extracted from the white seeds is
lower than in the black seeds, where the percentage was
2.82% and 3.15% respectively. By comparing the fatty
acids studied with the results of the research, we found
that the ratio of myristic acid is higher than in [13] where
it was 0.04% while [19] for this fatty acid (nonexistent)
while [16] said that the ratio of myristic acid was 0.07%, as
for the unsaturated fatty acid myristoleic (omiga -5),
which was diagnosed from the oil extracted from the white
seed did not indicate the existence of any of the research
above, as observed in table (2), we found that the
percentage of saturated fatty acid (palmitic) was 3.18%,
which is lower than that stated in [13] [19] and [16] with
7.47%, 7.1% and 7.07% respectively, while the ratio of olic
fatty acid was 1.79%. This result is similar to that of [13],
where it was 2.43%, while this result did not correspond
to [19] and [16] respectively, where it was 6.4% and
7.04%, respectively, and the ratio of linoleic acid was
2.97%, where it did not correspond to what is stated in
[13],[19] and [16] where it was 68.52%, 64% and 62.80%,
respectively, and the ratio of fatty acid Lignoceric was
0.69%, which is higher than indicated by [16] While the
percentage of Erucic fatty acid (omiga-9) was 3.3%, which
was diagnosed in the oil extracted from black seeds, where
not referred to Research from above.
Table-2: Fatty acids and their proportion of oil
extracted from white seeds
Fatty acids of white
seeds
Percentage of fatty
acids in oil %
Myristic C14:025.2
Myristoleic C14:1n-52522
Arachidic C20:03532
Linoleic n-6 C18:265..
Table-3: Fatty acids and their proportion of oil
extracted from black seeds
Fatty acids of black seedsPercentage of fatty
acids in oil %
Myristic C14:03.15
Palmitic C16:03.18
Oleic C18:1n-91.79
Linolenic C18:3n-32.97
Linoleic C18:2n-64
Erucic C22:1n-93.3
Lignoceric C24: 00.69
The difference in the percentage of fatty acids obtained in
comparison with other studies may be due to the
difference in the type of Chia seeds and to the surrounding
conditions in addition to the difference in the planting
location of the seeds and the difference of climate factors,
it may also be due to different extraction method and the
type of solvents used and the degree of purity as well as
some processes that may be performed on extracted oil
such as purification factors and may also be due to the
difference in the type and accuracy of the device in
addition to the circumstances surrounding the preparation
of the sample and the extent of experience of the person
existing this Operation. All of these factors lead to
divergent results.
Figure -1: Standard fatty acids

Figure -2: Fatty acids of white chia seed oil
Figure -3: Fatty acids of black chia seed oil
3.3 Active compounds
The results of the qualitative detection of some active
compounds in the extracted oil from white and black chia
seeds showed that the oil contained the tannins, terpenes
and flavonoids, as the results of the tests on these
compounds were positive, as shown in Table (4). These
results are consistent with the findings of [11][12][17],
which refers to the presence of secondary compounds,
tannins, terbines and flavonoids.
Table-4: Results of qualitative detection of active
compounds in chia seed oil
DetectionWhite chia seed oilblack chia seed oil
Type
Detector
Reagent AReagent BReagent AReagent B
terbines+Reddish
brown
+Brown
deposit
+Reddish
brown
+Brown
deposit
tannins+Bluish
green
+ Yellow
precipitate
+Bluish
green
+Yellow
precipitate
flavonoid
s
+Red
Orange
+Red+Red
Orange
+Red
*The signal + indicates the presence of the active
compound
3.4 Antioxidant efficacy
Table (5) shows the effect of the concentrations of oil
extracted from white chia seeds in the percentage of
antioxidant activity compared with Ascorbic Acid as a
control group. The percentage of inhibition was increased
to 88.37% at the concentration of 400μl/ml, 83.6%, 72.5
and 57.2 at concentrations (50, 100,200) μl/ml,
respectively. The results of the present study also showed
the percentage of antioxidant efficacy of oil extracted from
black chia seeds compared with Ascorbic Acid as a control
group. The percentage of inhibition was increased by
94.5% at 400 μl/ml, 86.38, 72.6 and 39.7 at concentrations
(50) (100,200) μl/ml, respectively, and this result is
consistent with what is indicated by [18]. The percentage
of inhibition of oil extracted from chia seeds may reach
more than 90% by using high concentration extracted oil.
This may be due to the high concentration of active
compounds, which may be attributed to the antioxidant
effect, as the oil contains many active compounds that have
been diagnosed as tanninates, turbines and flavonoids
where several studies have investigated its antioxidant
effectiveness, with high hydroxyl flavonoids and rutin
flavonide acting as antioxidants [22]. The difference in the
inhibition ratio between the two types of oil extracted
from the white and black seeds may be due to the
difference in concentration of these active compounds in
extracted oil. In addition, the oil may contain other
compounds found in the oil extracted from the black seeds
and are not present in the oil extracted from the white
seeds because of the different genetic makeup of the seeds.

Table-5: Comparison of extracted oil from Chia seeds
as an antioxidant with ascorbic acid
concentration
l µ/ ml
Inhibition
ratio of
white seed
oil
%
Inhibition
ratio of
black seed
oil
%
Comparative
model
%
.33..5689.5.9.53
233.653.356.9.52
233825.8253.653
.3.8526958.3539
2.225.22533.3533
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